Introduction The dynamic control of intracellular iron concentrations is paramount to all biological systems. One aspect of this issue is that biologically useful iron (i.e. Fe2") is extremely limiting or it is highly insoluble (i.e.Fe3). Accordingly, biological entities have evolved efficient mechanisms to acquire this nutrient from the insoluble form, which is generally in plentiful quantities. On the other hand, further acquisition of iron above biologically useful concentrations can have dire consequences for a cell. Excess free iron will catalyze the generation of highly reactive oxygen and nitrogen intermediates that will damage all known biological macro-molecules. This conflict, in a major way is dealt with in a diverse array of pathogenic microbes, by a classic Fe2+-dependent represser called Fur (ferric uptake regulator) that plays the key role in controlling iron homeostasis. While it is clear that the Class B Select Agents Burkholderia pseudomallei & mallei express a protein that is highly homologous to Fur from closely related organisms (e.g. Pseudomonas aeruginosa) very little is known about the role of iron homeostasis in the virulence of 8. pseudomallei & mallei. More importantly, nothing is known about the role of iron homeostasis in the intracellular life style of these pathogens. We propose to remedy this situation through the use of powerful molecular and genetic approaches that we employed to elucidate the extensive and crucial role that Fur plays in the related, but distinct, opportunistic pathogen P. aeruginosa. We will identify the Fur regulated genes of 6. pseudomallei & mallei that encode regulatory factors (e.g. sigma factors, small regulatory RNAs). We will then evaluate the role of these selected regulatory factors in the ability of these agents to enter, survive in, grow in or traffick in model eukaryotic cells (e.g. macrophage), which are appropriate to the known aspects of the pathogenesis of these potential bioterrorist agents. The gene products that we identify may ultimately be used in developing therapeutic and prophylactic agents against Malleipdosis and Glanders. Project interactions In addition to the obvious interactions with other project members (e.g. Drs. Vazquez- Torres, Holmes, & Voskuil) that are described in more detail in the body of this application and in the Introduction to Project II.C, we will especially interact with Dr. Robison (BYU Select Agent Archive) and Dr. Schweizer (Microbial genetics - CSU) in terms of the bacteriology and genetics of B. pseudomallei & mallei. We will also be especially interested in very active interactions with Dr. Slayden (Genomics/Proteomics Core) that will be vital to development of microarrays and for the evaluation of potential target genes and products for possible use in experimental prophylactic and therapeutic agents. Also, along these lines we will also closely interact with Dr. Brennan (PDM Core) for the same purpose.